Journal: eLife

Article Title: A conserved mycobacterial nucleomodulin hijacks the host COMPASS complex to reprogram pro-inflammatory transcription and promote intracellular survival

doi: 10.7554/eLife.107677

Figure Lengend Snippet: ( A ) Y2H assay identifying interactions between MgdE mutants and COMPASS complex subunits. Yeast cells were co-transformed with bait (pGBKT7) and prey (pGADT7) plasmids expressing wild-type or mutant MgdE and human COMPASS subunits (ASH2L, WDR5, RbBP5, and DPY30). Growth was assessed on non-selective (-Leu/-Trp, left) and selective (-Leu/-Trp/-Ade/-His+200 ng/μL aureobasidin A, right) media. Controls: CK− (pGBKT7- lam +pGADT7 T, negative) and CK+ (pGBKT7- p53 +pGADT7 T, positive). ( B ) Co-IP analysis of MgdE mutants with WDR5. HEK293T cells were co-transfected with Flag-tagged MgdE mutants and HA-tagged WDR5 (1:1 molar ratio). Complexes were immunoprecipitated using anti-HA antibody and protein A/G beads, followed by immunoblotting with anti-Flag and anti-HA antibodies. ( C ) Nuclear distribution of wild-type and mutants MgdE. Confocal microscopy of HEK293T cells expressing wild-type or D224AH247A MgdE-EGFP at 12 and 24 hr post-transfection (hpt). Nuclear foci were visualized by enhanced green fluorescent protein (EGFP) (green) and DAPI (blue) staining. Scale bar, 10 µm. Images were acquired with a 100x oil immersion objective (NA = 1.4). ( D ) Immunoblot analysis of H3K4me3 levels. HEK293T cells expressing wild-type or D224AH247A mutant MgdE were analyzed for changes in H3K4me3 levels over 0-24 hr post-transfection. Histone H3 was used as a loading control. Data represent mean ± SD of three independent biological replicates. Statistical significance was determined using two-tailed unpaired Student’s t -tests, * p <0.05, ** p <0.01, *** p <0.001. Figure 5—source data 1. Original files for western blot analysis displayed in panel B. Figure 5—source data 2. Original western blots for panel B, indicating the relevant bands. Figure 5—source data 3. Original files for western blot analysis displayed in panel D. Figure 5—source data 4. Original western blots for panel D, indicating the relevant bands.

Article Snippet: The Y2H Gold yeast strain (Takara Bio) was maintained in YPDA (yeast extract, peptone, dextrose, and adenine sulfate) medium.

Techniques: Y2H Assay, Transformation Assay, Expressing, Mutagenesis, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot, Confocal Microscopy, Staining, Control, Two Tailed Test

Journal: Veterinary Microbiology

Article Title: EIF3i affects vesicular stomatitis virus growth by interacting with matrix protein

doi: 10.1016/j.vetmic.2017.10.021

Figure Lengend Snippet: VSV M protein interacts with the i subunit of eIF3. (A) Binding of the VSV-M protein to eIF3i in a yeast two-hybrid system. Yeast strain AH109 was co-transformed with a bait plasmid (BD-M) and a prey plasmid (AD-eIF3i), which encodes the full-length eIF3i fused to the GAL4 activation domain. The empty vector BD was co-transformed with AD-eIF3i to exclude self-activation. Co-transformation of BD-Lambda/AD-T, BD-p53/AD-T and BD/AD were used as negative, positive and blank controls, respectively. (B) GST-M pull-down assay. The GST and GST-M proteins expressed in E. coli BL21 (DE3) were purified with glutathione Sepharose 4B beads (catalog no. 17075601; GE Healthcare). The beads conjugated with GST or GST-M were incubated with the recombinant Flag-eIF3i protein. After washing with cold PBS, the bound proteins were separated by SDS-PAGE (12%) and detected by western blotting. (C) GST- eIF3i pull-down assay. The GST or GST-eIF3i proteins expressed in E. coli BL21 (DE3) were purified with glutathione Sepharose 4B beads (catalog no.17075601; GE Healthcare). The beads conjugated with GST or GST-eIF3i proteins were incubated with the recombinant Flag-M protein. (D) Co-localization of M with eIF3i in the cells. The cells were co-transfected with pT-EGFP-M (or pT-EGFP, as control) and pT-DsRed-eIF3i (or pT-DsRed, as control) and fixed with 4% paraformaldehyde at 24 h post-transfection. The results were obtained with a laser confocal scanning microscope. All the experiments were repeated three times. SD/-2, SD/-Leu/-Trp; SD/-4, SD/-Ade/-His/-Leu/-Trp.

Article Snippet: The normalized mouse brain cDNA library fused to the sequence encoding the GAL4 activation domain, pre-transformed in the Y187 yeast strain, was purchased from Clontech (catalog no. 630488; Clontech).

Techniques: Binding Assay, Transformation Assay, Plasmid Preparation, Activation Assay, Pull Down Assay, Purification, Incubation, Recombinant, SDS Page, Western Blot, Transfection, Control, Microscopy